Parallel Peptide Tandem Mass Spectrometry (MS)

描述(由申请人提供)：这是5R33CA099139-04并行多肽串联质谱学的修订申请，该计划NOT-OD-09-058 NIH宣布恢复法案资金可用于竞争性修订申请。最初的R33奖是围绕一种方法设计的，该方法通过将存在于较宽(例如400-1400)m/z范围内的所有多肽碎裂来增加蛋白质识别。其想法是，这种猎枪CID(或多路碰撞诱导解离)比标准的猎枪蛋白质组学方法更有效，标准的猎枪蛋白质组方法在将样品引入串联质谱仪期间使用依赖于DAD的离子选择。虽然这种方法奏效了，但效率被许多标识中的退化所抵消。这导致我们修改了基本的数据获取方法。我们不是在一个宽的m/z范围内查看所有的多肽，而是进行了一个实验来查看每个m/z“通道”中的所有多肽。显然，这需要更多的注射，但如下所述，该方法非常有效地增加了有信心的蛋白质鉴定的数量、蛋白质序列覆盖率和动态范围，所有这些都是在没有事先进行样品分离的情况下实现的。缺点是获取数据集所需的时间，通常是连续5天的自动化LC-MS/MS时间。在此，我们请求将我们的R33延长一年，以优化我们称为独立于离子计数(Pacific)的多肽采集方法：目标1)优化独立于离子计数(Pacific)的多肽采集，使其定量和多路传输，并将采集时间减少到两天；目标2)使用前体离子独立自顶向下算法(PITA)通过修改湿法和干法实验室方法来识别“小”蛋白质，以及结合Pacific和Aim 3)识别和消除化学噪声源，在每个m/z通道上系统地获取太平洋数据集，可以透彻地了解所有可检测到的多肽/蛋白质(从低和高信号/噪声中)，以及如何减少非肽“噪音”的来源，以帮助检测低丰度蛋白质。 与公共健康相关：许多NIH项目的一个统一目标是大幅减少癌症死亡。目前的想法是，这可能是通过及早检测疾病的标志来实现的，例如蛋白质和代谢物。支持我们建议的初步数据显示，通过对获取和分析蛋白质组数据的方式进行简单修改，可以识别新的蛋白质。我们提议的计划将带来更简便的蛋白质发现方法，这些方法可能会立即被寻找疾病蛋白质标记物的实验室采用。

DESCRIPTION (provided by applicant): This is a revision application of 5R33CA099139-04 Parallel Peptide Tandem Mass Spectrometry to the program NOT-OD-09-058 NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The original R33 award was devised around a method to increase protein identifications by fragmenting all peptides present in a wide (e.g. 400-1400) m/z range. The idea being that this shotgun CID (or multiplexed collision induced dissociation is more efficient than standard shotgun proteomic methods that use dad-dependent ion selection during HPLC introduction of the sample to a tandem mass spectrometer. While this approach worked, the efficiency was offset by the degeneracy in many of the identifications. This led us to modify our basic data acquisition approach. Instead of looking at all peptides in a wide m/z range, we carried out an experiment to look at all peptides in each m/z "channel". Obviously, this requires many more injections, but as described below the approach worked remarkably well to increase the number of confident protein identifications, protein sequence coverage and dynamic range all of which was achieved without prior sample fractionation. The downside is the time it takes to acquire data set, typically five days of continuous automated LC-MS/MS time. Here we request a one year extension of our R33 to optimize a method we refer to as Peptide Acquisition Independent From Ion Count (PAcIFIC) by: Aim 1) Optimize Peptide Acquisition Independent From Ion Count (PAcIFIC) making it quantitative and multiplexed as well as reducing acquisition time to two days, Aim 2) Use Precursor Ion Independent Top- down Algorithm (PIITA) to identify "small" proteins by modification of wet and dry lab methods as well as to incorporate PAcIFIC and Aim 3) Identify and eliminate sources of chemical noise, PAcIFIC data sets being systematically acquired on each and every m/z channel allows a thorough understanding of all peptides/proteins detectable (from low and high signal/noise) and how sources of non-peptide "noise" may be diminished to aid detection of low abundance proteins. PUBLIC HEALTH RELEVANCE: A unifying goal of many NIH programs is to drastically reduce deaths from cancer. The current thinking is this may be achieved through early detection of markers of disease; e.g. proteins and metabolites. Preliminary data supporting our proposal shows that new proteins may be identified by simple modifications to the way proteomic data is acquired and analyzed. Our proposed plan will lead to more facile methods for protein discovery that may be immediately adopted by laboratories searching for protein markers of disease.